title: A southern blot assay for detection of hepatitis B virus covalently closed circular DNA from cell cultures.
abstract: Chronic hepatitis B remains a substantial public health burden affecting approximately 350 million people worldwide, causing cirrhosis and liver cancer, and about 1 million people die each year from hepatitis B and its complications. Hepatitis B is caused by hepatitis B virus (HBV) infection. As an essential component of the viral life cycle, HBV covalently closed circular DNA (cccDNA) is synthesized and maintained at low copy numbers in the nucleus of infected hepatocytes, and serves as the transcription template for all viral RNAs. Therefore, cccDNA is responsible for the establishment of viral infection and persistence. The presence and longevity of cccDNA may also explain the limitations of current antiviral therapy for hepatitis B. Thus, understanding the mechanisms underlying cccDNA formation and regulation is critical in understanding the HBV pathogenesis and finding a cure for hepatitis B. Here we describe a protocol for HBV cccDNA extraction and detection in detail. The procedure includes two major steps: (1) HBV cccDNA extraction by Hirt protein-free DNA extraction method and (2) HBV cccDNA detection by Southern blot analysis. The method is straightforward and reliable for cccDNA assay with cell culture samples, and it is useful for both HBV molecular biology and antiviral research.
authors.affiliation: Institute for Biotechnology and Virology Research, Drexel University College of Medicine, Doylestown, PA, USA.
authors.forename: Dawei
authors.initials: D
authors.lastname: Cai
authors.forename: Hui
authors.initials: H
authors.lastname: Nie
authors.forename: Ran
authors.initials: R
authors.lastname: Yan
authors.forename: Ju-Tao
authors.initials: JT
authors.lastname: Guo
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Block
authors.forename: Haitao
authors.initials: H
authors.lastname: Guo
mesh_terms: D015139:Blotting, Southern / Q000379:methods; D018929:Cell Culture Techniques; D002460:Cell Line; D004270:DNA, Circular / Q000302:isolation & purification*; D004279:DNA, Viral / Q000302:isolation & purification*; D056945:Hep G2 Cells; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans
chemical_list: D004270:DNA, Circular; D004279:DNA, Viral